本文選題：大鯢 + β-defensin基因��； 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：β-defensin是一種結(jié)構(gòu)穩(wěn)定,抗菌抗病毒活性強(qiáng),具備多種生物學(xué)活性的小分子多肽。本研究對來自于大鯢皮膚轉(zhuǎn)錄組數(shù)據(jù)庫中的大鯢β-defensin基因進(jìn)行了鑒定和結(jié)構(gòu)分析;采用熒光實(shí)時(shí)定量PCR檢測了大鯢β-defensin基因在不同組織、不同發(fā)育階段以及細(xì)菌和病毒感染情況下的表達(dá)特性和變化;并對大鯢β-defensin基因進(jìn)行了重組原核表達(dá)載體的構(gòu)建和重組蛋白活性的檢測。獲得如下結(jié)果:1.大鯢β-defensin基因全長cDNA為771 bp,其中5’非編碼區(qū)為90bp,3’非編碼區(qū)為531 bp,開放閱讀框150bp,共編碼49個(gè)氨基酸,編碼的蛋白質(zhì)分子量為5.52 kD,等電點(diǎn)為9.23,在pH等于7的溶液中的電荷為5.87,無信號肽,為內(nèi)源性抗菌肽。基于氨基酸序列的同源聚類關(guān)系表明,其和兩棲動(dòng)物親緣關(guān)系最近。2.實(shí)時(shí)熒光定量PCR表明,大鯢在皮膚中表達(dá)量最高,且在幼體時(shí)即大量表達(dá)。細(xì)菌和病毒感染會(huì)顯著上調(diào)大鯢β-defensin基因的表達(dá),其中細(xì)菌感染后大鯢β-defensin基因在24小時(shí)達(dá)到峰值,而病毒感染后則在120小時(shí)達(dá)到峰值。3.構(gòu)建獲得的大鯢重組表達(dá)載體pET32a(+)-AdBD經(jīng)IPTG誘導(dǎo),獲得了大鯢重組β-防御素蛋白,純化后的重組蛋白對革蘭氏陰性菌和革蘭氏陽性均有殺滅效果,其中對革蘭氏陰性菌中的溶藻弧菌、大腸桿菌和哈維氏菌最有效,最小抑菌濃度僅為19μg/mL。以上結(jié)果表明大鯢β-defensin基因是大鯢皮膚主要免疫效應(yīng)基因,對細(xì)菌和病毒感染均有積極響應(yīng),其中對細(xì)菌感染響應(yīng)迅速。同時(shí)重組原核表達(dá)的大鯢β-defensin蛋白對多種細(xì)菌均有很好的抗菌活性,是生物活性肽資源開發(fā)的潛在對象。
[Abstract]:尾 -defensin is a small polypeptide with stable structure, strong antibacterial and antiviral activity and various biological activities. In this study, the 尾 -defensin gene from the skin transcriptional database of giant salamander was identified and its structure was analyzed, and the 尾 -defensin gene in different tissues was detected by real-time fluorescence quantitative PCR. The expression characteristics and changes of 尾 -defensin gene in different stages of development and bacterial and viral infection were studied, and the recombinant prokaryotic expression vector was constructed and the activity of recombinant protein was detected. Get the following result: 1. The full-length cDNA of the 尾 -defensin gene of giant salamander is 771bp, of which the 5'noncoding region is 90bpf3'and the open reading frame is 150bp. it encodes 49 amino acids. The encoded protein has a molecular weight of 5.52 KD, a isoelectric point of 9.23 and a charge of 5.87 in a solution with pH = 7. It is an endogenous antimicrobial peptide. The homologous clustering relationship based on amino acid sequence shows that it has the closest genetic relationship with amphibians. Real-time fluorescence quantitative PCR showed that Giandrias davidianus had the highest expression in skin and was highly expressed in larvae. Bacterial and viral infection significantly up-regulated the expression of 尾 -defensin gene. The peak of 尾 -defensin gene reached a peak at 24 hours after bacterial infection, and reached a peak value at 120 hours after viral infection. The recombinant expression vector pET32a was constructed. The recombinant 尾 -defensin protein of salamander was induced by IPTG. The purified recombinant protein could kill both Gram-negative bacteria and Gram-positive bacteria. Among them, vibrio alginolyticus, Escherichia coli and Harvey's bacteria in Gram-negative bacteria were the most effective, the minimum inhibitory concentration was only 19 渭 g / mL. The results showed that the 尾 -defensin gene was the main immune effector gene in the skin of giant salamander, and had a positive response to both bacterial and viral infection, and the response to bacterial infection was rapid. At the same time, the recombinant 尾 -defensin protein of giant salamander has good antibacterial activity against a variety of bacteria, and it is a potential target for the exploitation of bioactive peptide resources.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S917.4;S947.3

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